4-Me-1-(2-(1H-tetrazol-5-yl)ethyl)benzene sulfonate and N-2-(1H-tetrazol-5-yl)ethyl methanesulfonamide

ABSTRACT

At least one compound defined by one of the following formulae: ##STR1## where X represents Cl, Br, ##STR2## or--SCH 3 , and n is 1 or 2; and 
     
         CH.sub.3 SO.sub.2 NHCH.sub.2 CH.sub.2 CN                   (II) 
    
     have been administered to animals to improve the efficiency of food utilization by these animals. These compounds can be administered orally by combining the compounds with feed compositions and fed to animals such as ruminants with a developed rumen function as well as other animals that ferment fibrous material and vegetable matter in the cecum or colon. These compounds effect the rumen metabolism to increase the production of propionate relative to other volatile fatty acids, particularly acetate and to inhibit methanogenesis.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

This invention relates to novel compounds and methods for increasing theefficiency of feed utilization in animals, particularly ruminantanimals.

More specifically, this invention relates to feed additives for ruminantfeed compositions which alter rumen metabolism to result in inhibitionof methane production and an increase in propionate production relativeto acetate.

(2) State of the Art

The improvement of feed efficiency of ruminant animals has for manyyears been a highly desirable commercial objective. The economicincentive is clear. If a method is devised for increasing feedefficiency and/or the rate of weight gain of cattle or sheep whiledecreasing the amount of feed consumed by these animals, then thefarmer's costs for raising these animals are reduced.

In order to improve the efficiency of feed utilization by ruminants,first it was necessary to discover and understand the mechanism by whichthese ruminants digest the various components of their feed and todetermine the resultant digestion products which are metabolicallyutilized by the animal. As a result of extensive work in this area, themechanism for carbohydrate utilization by ruminant animals is well knownand documented in the literature. Raun in his U.S. Pat. Nos. 3,790,667,3,790,668, 3,794,732 and 3,839,557, the disclosures of same are hereinincorporated by reference, clearly sets out what is recognized as theaccepted mechanism for the utilization of the feed carbohydrates byruminant animals and further discloses the relevant references teachingthe early work conducted in this area of technology. Raun found thatpropionate production could be increased and in turn improve ruminantfeed utilization by orally administering to the ruminants selectedpolyether antibiotics and such physiologically-acceptable compounds, asthiostrepton, monensin and dianemycin. It is also pointed out in theRaun patents that inducing an increase in propionate production in therumen of the animal results in a secondary benefit apart from theincrease in feed utilization efficiency. The secondary benefit being theinhibition of ketosis which is the result of a high proportion ofacetate in the rumen and which amounts to a clinical illness in theanimal.

Since Raun's work, further studies have been conducted investigating theeffect of monensin, in particular, on rumen metabolism, e.g., Van Nevel,et al., Appld. and Envl. Microbio., 34(3), pgs. 251-257 (Sept. 1977) andSlyter, Appld. and Envl. Microbio., 37(2), pgs. 283-288 (Feb. 1979). Inaddition, others have found that such additives as polyhalohemiacetalderivatives of saccharides and polysaccharides, Parish, et al., U.S.Pat. No. 3,615,649 and polyhaloalkamines, Parish, et al., U.S. Pat. No.3,733,417, can improve the feed efficiency of ruminant animals. None ofthese feed additives, however, have proven to achieve the optimumresults with respect to preventing the animal from reducing feed intakewhile at the same time inducing the optimal increase in propionateproduction in the rumen of these animals over an extended period oftime.

SUMMARY OF THE INVENTION

It has now been discovered, in accordance with the present invention, anovel method for regulating rumen metabolism over an extended period oftime.

Further in accordance with the present invention, novel compounds havebeen prepared utilizing an efficient mode of preparation.

Still further in accordance with the invention, specific 5-substitutedtetrazoles and a precursor demonstrate the ability to regulate themetabolism of rumen microorganisms. More specifically, these compoundsare shown to effect the inhibition of methanogenesis, to increase rumenpropionate concentration, to decrease the rumen acetate concentrationand to increase fermentation efficiency in ruminant animals.

These and other aspects of the invention will become clear to thoseskilled in the art upon the reading and understanding of thespecification.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

It has been recognized for several years that if methanogenesis could bepartially or totally inhibited with a corresponding increase inpropionate production in the rumen, the result would be thatapproximately 8 percent of the feed energy which is normally lost(ultimately through eructation) would be made available to the growinganimal, see, for example, Hungate, The Rumen And Its Microbes, AcademicPress, N.Y. (1966), pp. 246 and 247. Diversion of this energy loss frommethanogenesis would in turn make this energy available for moreproductive metabolic processes such as the biosynthesis of propionatewhich is a major source of metabolic energy. The overall result for theanimal might be expressed as improvement of feed efficiency andincreased rate of weight gain. Thus, discovering the means for themetabolic regulation of methanogenesis would be a productive approach toultimately improving the efficiency of feed utilization for ruminantanimals.

It was found through extensive in vitro and in vivo studies thatspecific 5-substituted tetrazoles of formula I below and a precursorcompound of formula II below, demonstrated the ability to inhibitmethanogenesis and increase propionate production relative to acetate inthe rumen of the animals. ##STR3## where X is --Cl, --Br, ##STR4## or--SCH₃, and n is 1 or 2; and

    CNCH.sub.2 CH.sub.2 NHSO.sub.2 CH.sub.3                    (II)

The 5-substituted tetrazoles of the present invention are prepared bydirect, efficient and high yield synthetic techniques, this being one ofthe advantages of the compounds of the present invention over othercompounds disclosed in the prior art. The 5-substituted tetrazoles ofthis invention are generally prepared according to the following scheme:##STR5## where X and n are the same as previously defined herein.

The different azide reagents which can be used are (1) NaN₃ +NH₄ Cl inDMF as disclosed in U.S. Pat. No. 2,977,372; (2) AlCl₃ +NaN₃ in THF asdisclosed in J.O.C., 34(4), (1969) 1141-1142; and (3) (n-Bu)₃ SnN₃ intoluene as disclosed in J. Organomet. Chem., 33 (1971) 337-346. Theselection of the particular azide reagent will be dependent upon thereaction conditions required to prepare the specific 5-substitutedtetrazole, cost, availability of materials as well as other suchconsiderations. It has been found, however, that while all of thesereagents generally lead to the desired products, reagent (1) suffersfrom the difficulty of complete removal of DMF; reagent (2) was found tobe useful in the preparation of water insoluble tetrazoles; whilereagent (3) was found to be useful in preparing tetrazoles whichrequired mild reaction conditions such as the chloroacetamido species.The alkylene nitrile reactants or precursors for the preparation of the5-substituted tetrazoles of the present invention can be purchasedthrough most bulk chemical suppliers. The particular procedure followedfor preparing the 5-substituted tetrazoles of the present inventioncomprised heating the mixture of reactants and the selected azidereagent in the selected solvent for a period of time of reflux. Whilethe actual reaction time is not critical, it was found the best resultswere achieved if the reaction took place for a minimum of 12 hours.Specific preparative procedures for compounds within the scope of thisinvention will be further illustrated in the examples that follow thisdiscussion.

The in vitro as well as the in vivo studies conducted show the compoundsof the present invention to be effective for inhibiting methaneproduction by ruminant animals as well as increasing the relativepropionate concentration in the rumen. It was also discovered that whilethe compounds of this invention were quite effective for the disclosedutility, their effectiveness can be enhanced by mixing the compounds ofthis invention with other known rumen metabolic regulators, e.g.,polyether antibiotics (monensin, etc.). The relative amounts of thesecompounds administered were not found to be critical except as isevident, the amounts must be kept below toxic levels.

Various features and aspects of the present invention will be furtherillustrated in the examples that follow. While these examples will showone skilled in the art how to operate within the scope of thisinvention, they are not to serve as a limitation on the scope of theinvention where such scope is only defined in the claims.

COMPOUND PREPARATION

The following examples will serve to illustrate the synthesis of thecompounds within the scope of this invention.

EXAMPLE 1 Preparation of 5-(2-Chloroethyl)Tetrazole

A mixture of 39.9 g (0.3 mol) of AlCl₃, 600 mL of THF, and 86.78 g (1.33mol) of NaN₃ and 26.86 g (0.3 mol) of ClCH₂ CH₂ CN were stirred andheated to reflux under Argon for 24 hours then cooled and acidified with450 ml of 15 percent HCl solution. The resulting mixture was warmed on asteam bath to remove NH₃. The phases were then separated and the aqueousphase was extracted twice with 150 mL portions of ethyl acetate. Theorganic phases were combined, washed with saturated NaCl solutionfollowed by drying over anhydrous Na₂ SO₄. Removal of the solvent underreduced pressure gave 33.87 g of a brown solid, m.p. 88°-102.5° C. Threerecrystallizations from ClCH₂ CH₂ Cl gave 23.55 g (59 percent) ofdesired compound as fine beige needles, m.p. 104°-105° C.

EXAMPLE 2 Preparation of 5-Chloromethyltetrazole

To a mixture of 26.6 g (0.2 mol) of AlCl₃ and 450 mL dry THF was addedwith stirring and in one portion, 57.2 g (0.88 mol) of NaN₃. Theresulting mixture was stirred under Argon at room temperature for 15minutes followed by the addition of 15.1 g (0.2 mol) of ClCH₂ CN. Theresulting mixture was heated and stirred at reflux for 24 hours thenquenched with 300 mL of 15 percent HCl solution. The resulting 2-phasemixture was heated on a steam bath for 15 minutes to remove HN₃ afterwhich the phases were separated. The aqueous phase was washed 3 timeswith 100 mL portions of ethyl acetate and then dried over anhydrous Na₂SO₄. Removal of the solvent under reduced pressure gave 26.07 g of blackwax which was dissolved in 25 mL of H₂ O. The resulting solution wasadjusted to a pH 10 with NaOH. The aqueous alkaline solution wasextracted 3 times with 100 mL portions of ether. The aqueous phase wasthen adjusted to pH 2 with 15 percent HCl solution. The aqueous phasewas saturated with NaCl then extracted 3 times with 100 mL of ethylacetate. The ethyl acetate phases were combined, washed with brine andthen dried over anhydrous Na₂ SO₄. Removal of solvent under reducedpressure gave 17 g of brown solid which was recrystallized from ClCH₂CH₂ Cl to give 9.74 g of a brown solid, m.p. 72°-79° C. This brown solidwas in turn recrystallized from CHCl₃ to give 5.33 g of the desiredcompound, m.p. 88.5-90.5. The mother liquor afforded an additional 0.98g of the desired compound, m.p. 87°-88° C.

EXAMPLE 3 Preparation of 2-chloro-N-(2-(1H-Tetrazol-5-yl)ethyl)acetamide

A solution of 9.3 g (65 mmols) of ##STR6## and 32.37 g (97.5 mmols) of(n-Bu)₃ SnN₃ in 150 mL of toluene was stirred and heated at reflux for19 hours then cooled to room temperature. The supernatant liquid wasdecanted from the brown oil which formed. Removal of the solvent underreduced pressure gave a cloudy oil-solid mixture. The material wasvigorously stirred with 300 mL of ether while anhydrous HCl gas waspassed through the mixture resulting in a wax. The ether was decantedafter which the wax was dissolved in 300 mL of hot ethyl acetate andanhydrous HCl gas was passed through the resulting solution. Thesolution became cloudy and an oil formed. The supernatant was separatedfrom the oil and the solvent was removed from the supernatant underreduced pressure to give 2.61 g of an oily solid. This material waswashed by stirring with ether then filtered to give 1.86 g of beigesolid, m.p. 105°-120° C. Three recrystallizations of this material fromabsolute ethanol afforded 0.658 g of the desired compound as colorlessto white nodules, m.p. 132°-133.5° C.

EXAMPLE 4 Preparation of4-methyl-1-(2-(1H-Tetrazol-5-yl)ethyl)benzenesulfonate

A solution of 11.26 g (50 mmols) of ##STR7## 19.92 g (60 mmols) of(n-Bu)₃ SnN₃, and 150 mL of dry THF was stirred and heated at reflux for24 hours then cooled to room temperature. Removal of the solvent underreduced pressure gave a clear pale yellow oil which was stirred at roomtemperature for 30 minutes with 350 mL of ethereal HCl solution. Theresulting mixture was filtered followed by washing the solid by stirringfor 2 hours with 200 mL of ether to give 4.03 g of white powder, m.p.150.5°-152.5° C. Recrystallization from 135 mL of boiling ethyl acetateafforded 2.98 g of the desired compound as colorless needles, m.p.158°-159° C.

All the 5-substituted tetrazoles within the scope of this invention areprepared by the procedures illustrated in the previous examples. Asindicated previously, the preparation of these compounds is direct andefficient, giving good yields of the desired compound.

IN VITRO STUDIES A. Procedure

Below is set forth the procedure followed for testing the compounds ofthe present invention in vitro. For these studies, rumen fistulatedsheep were fed a concentrate diet which constituted 3 parts chopped cornand cob and 1 part alfalfa pellets.

1. Rumen fluid was removed from a fistulated sheep and strained through4 layers of cheese cloth to remove large feed particles.

2. One part rumen fluid was added to 3 parts of modified McDougal'sArtificial Saliva, the pH was adjusted to 7.0 and the saliva solutionwas prewarmed to 39° C. in a water bath and gassed with CO₂.

(a) Modified McDougal Artificial Saliva composition:

    ______________________________________                                        Salt             mmoles                                                       ______________________________________                                        NaHCO.sub.3      58.5                                                         KHCO.sub.3       58.5                                                         NaH.sub.2 PO.sub.4                                                                             13.0                                                         KH.sub.2 PO.sub.4                                                                              13.0                                                         NaCl             8.0                                                          KCl              8.0                                                          MgCl.sub.2       0.3                                                          CaCl.sub.2       0.2                                                          ______________________________________                                        100 × salt solution                                                     Salt             g/L H.sub.2 O                                                ______________________________________                                        NaCl             47                                                           MgCl.sub.2       6                                                            KCl              57                                                           CaCl.sub.2       4                                                            ______________________________________                                    

To prepare (a), (i) 9.8 g NaHCO₃, 11.7 g KHCO₃, 5.1 g NaH₂ PO₄ and 4.08g KH₂ PO₄ were diluted with two (2) liters of distilled H₂ O; (ii) 20 mLof 100X salt solution was diluted to 1.33 l with tap H₂ O; and (iii) mix(i) with (ii) to prepare the modified McDougal Artificial Saliva.

3. Add 0.01 g concentrate feed/l mL of buffered rumen fluid (thebuffered rumen fluid with concentrate should be stirred at all times toprevent the concentrate from settling).

4. Dispense 20 mL of buffered rumen fluid into 30 mL serum bottles thatare being gassed with CO₂. (A manifold that gases 12 bottles at a timewas used.) The bottles should be in a 39° C. H₂ O bath.

5. The test compounds, i.e., the compounds of the present invention,were first added to the bottles. After the addition of the bufferedrumen fluid to all 12 bottles, the gassing cannulae were quickly removedand a butyl rubber stopper was inserted in each bottle. An aluminum sealwas then placed on each bottle and crimped tight. Note: do not removethe bottle from the water bath until it has been crimped.

6. The sealed bottles were removed from the water bath, shaken andplaced in a 39° C. incubator for 18 hours.

7. After the 18-hour incubation, the bottles were removed from theincubator and the head space gas was analyzed using a gas partitioner.

8. Four mL of rumen fluid was removed from each bottle for volatilefatty acid (VFA) analysis.

(a) The 4 mL sample was added to a centrifuge tube to which hadpreviously been added 0.04 mL of 50 percent H₂ SO₄ and 1 mL2-ethylbutyric acid (concentration 2 mL of 2-ethylbutyric acid/500 mL H₂O v/v).

(b) The mixture from (a) was centrifuged at 10000 xg for 20 minutes.

(c) The supernatant was removed and analyzed by gas-liquidchromatography. The above procedures were followed in all trials fortesting the compounds of the present invention.

B. Results

The results of the in vitro tests are out in Table I. It is pointed outthat these results are reported in terms of relative concentration ofthe volatile fatty acids (VFA) acetate, propionate, and butyrate andpercent methane inhibition to a negative control.

                                      TABLE I                                     __________________________________________________________________________                          Ratio Treated to Control                                                  Levels       Total                                                                             Methane                                    Compound          ppm C2.sup.2                                                                         C3.sup.2                                                                         C4.sup.2                                                                         VFA Inhibition (%)                             __________________________________________________________________________    .sup.1 TCH.sub.2 Cl                                                                             50.0                                                                              0.74                                                                             1.49                                                                             0.95                                                                             0.96                                                                              96.8                                       TCH.sub.2 CH.sub.2 Cl                                                                           50.0                                                                              0.76                                                                             1.48                                                                             0.92                                                                             1.03                                                                              74.6                                       TCH.sub.2 CH.sub.2 Br                                                                           50.0                                                                              0.73                                                                             1.44                                                                             1.25                                                                             0.91                                                                              91.3                                        ##STR8##         50.00                                                                             0.91                                                                             1.16                                                                             1.05                                                                             1.02                                                                              4.9                                        TCH.sub.2 CH.sub.2 NHSO.sub.2 CH.sub.3                                                          50.0                                                                              0.99                                                                             1.04                                                                             0.99                                                                             1.02                                                                              0.7                                         ##STR9##         50.0                                                                              0.92                                                                             1.23                                                                             0.95                                                                             1.07                                                                              8.0                                        CNCH.sub.2 CH.sub.2 NHSO.sub.2 CH.sub.3                                                         50.0                                                                              0.7                                                                              1.58                                                                             1.13                                                                             0.97                                                                              73.4                                       __________________________________________________________________________     ##STR10##                                                                     -                                                                             .sup.2 C2 represents acetate, C3 represents propionate and C4 represents      butyrate.                                                                

The results of the in vitro testing confirm that the compounds of thepresent invention are effective for increasing propionate productionrelative to acetate in rumen fluid as well as inhibiting the productionof methane.

IN VIVO STUDIES

The in vitro studies showed the compounds of the present invention hadthe desired effect on the biochemistry of the rumen bacteria, however,it had yet to be determined whether or not this effect would besustained in the animal where numerous other biochemical processes aresimultaneously occuring.

A. Procedure and Treatments

Eight rumen fistulated sheep were used as test animals. Two sheep werealotted to each of three treatments and a control for each test. Group Iwas always the control group in each of the different trials. Groups IIthrough IV had varying concentrations of compounds of the presentinvention added to the animals' feed. The feed ration in each trialconstituted 3 parts chopped corn and cob and 1 part alfalfa pellets.Each sheep was offered 800 g daily of the feed in two equal portions.The particular compound and vitamin premix were mixed with chopped cornand cob. Any feed refusals were weighed back before each feeding anddiscarded. To prevent any mold growth, the feed was stored underrefrigeration.

All animals in the various treatment groups were sampled for VFA's twicea week. Equal volumes of rumen fluid from each animal within a treatmentwere pooled for treatment VFA analysis. Fluid from individual animalswas also analyzed for VFA. Individual animal VFA's within a treatmentwere averaged. This value was then averaged with pooled treatment VFAyielding the final treatment VFA value. All ratios and statistics werecalculated using this final value. VFA's were calculated as the ratiotreated to its respective pretreatment value. Each of the trials lastedfor 90 days.

B. Results

Using the procedures set out in A, the data reported in Tables II andIII were collected from trials using two of the compounds of the presentinvention.

                  TABLE II                                                        ______________________________________                                        Compound: 5-Chloromethyltetrazole                                                          Ratio of Treatment                                                            VFA to Pretreatment VFA                                                  Level.sup.1                    Total                                  Groups  %          C2.sup.2                                                                              C3.sup.2                                                                             C4.sup.2                                                                           VFA                                    ______________________________________                                        I       Control    1.02.sup.3                                                                            0.99   0.90 0.87                                   II      0.01       1.01    0.98   1.02 0.96                                   III     0.05       0.97    1.05   1.04 0.88                                   IV      0.025      0.96    1.10   1.05 0.93                                   ______________________________________                                         .sup.1 Level is reported as percent of compound added to total amount of      feed administered to the animal. A dose level of 0.05 corresponds to          approximately 60 ppm of the compound in the rumen.                            .sup.2 C2 represents acetate, C3 represents propionate and C4 represents      butyrate.                                                                     .sup.3 Average of all values for biweekly samples taken over the 90day        trial period.                                                            

                  TABLE III                                                       ______________________________________                                        Compound: 5-(2-Chloroethyl)Tetrazole                                                       Ratio of Treatment                                                            VFA to Pretreatment VFA                                                  Level.sup.1                    Total                                  Groups  %          C2.sup.2                                                                              C3.sup.2                                                                             C4.sup.2                                                                           VFA                                    ______________________________________                                        I       Control    1.00.sup.3                                                                            1.05   1.07 1.00                                   II      0.05       0.97    1.06   1.11 0.88                                   III     0.075      0.92    1.24   1.05 0.89                                   IV      0.025      0.97    1.00   1.20 1.05                                   ______________________________________                                         .sup.1 Level is reported as percent of compound added to the total feed       administered to the animal. A dose level of 0.05 percent corresponds          approximately to 60 ppm of compound in the rumen.                             .sup.2 C2 represents acetate, C3 represents propionate and C4 represents      butyrate.                                                                     .sup.3 Average of all values for biweekly samples taken over the 90day        trial period.                                                            

As illustrated by the results of these trials, the compounds of thepresent invention were found to be effective in decreasing the acetateconcentration and correspondingly increasing the propionate productionin the rumen of the animals for a sustained time period.

Testing of other compounds within the scope of this invention givesresults that correspond to those set out in Tables II and III. It wasalso discovered that combining the compounds of the present inventionwith known rumen metabolic regulators, e.g., polyether antibiotics(monensin, etc.) resulted in a still further increase in propionateproduction with a concomitant decrease by both acetate and butyrate.Therefore, mixtures of two or more compounds of the present invention asfeed additives, as well as mixtures of compounds of the presentinvention with known rumen metabolic regulators are within the scope ofthis invention.

It has been found that the compounds of the present invention improvethe efficiency of feed utilization in ruminant animals when they areadministered orally to the animals. The simplest and easiest manner toorally administer the compounds of this invention to the animals is byadmixture to their feed. Any appropriate feed material for ruminantanimals may be used including the concentrate feed previously describedas well as roughage feeds such as silage or various commercial grainmixtures commonly used for ruminant animals.

The compounds of the present invention can be added to any conventionalpremix formulation, animal feed carries or adjuvants in an amountsufficient to increase the efficiency of feed utilization by ruminantanimals. In addition to the compounds of the present invention, theanimal feed compositions may contain such additives as vitamins;minerals; natural oils; e.g., vegetable oil, animal fat, fish oils,etc.; antioxidants; antibiotics; anthelmintics; and other appropriatemedicaments.

The compounds of the present invention may be administered to theanimals in other ways. For example, they may be incorporated intotablets, drenches, boluses, or capsules, and dosed to the animals informulations and by means well known in the veterinary pharmaceuticalart. They can also be administered in the field by means of salt ormolasses blocks. Use of the compounds of the present invention forimproving the efficiency of feed utilization of monogastric animalswhich digest at least a portion of their food by cecal and/or colonfermentation, since it follows a chemical pathway similar to rumenfermentation, is also contemplated by this invention.

In general, the scope of the present invention is not to be limited byany specific method of administration or any particular formulation ofthe compounds of this invention. Any manner or form for increasing theefficiency of feed utilization by ruminant animals by use of thecompounds of the present invention is within the scope of thisinvention.

Other features and aspects of this invention will be appreciated bythose skilled in the art upon reading and comprehending this disclosure.Such features, aspects and expected variations and modifications of thereported results are clearly within the scope of this invention wherethe invention is limited solely by the scope of the following claims.

What is claimed is:
 1. A compound selected from the group consisting of:##STR11## where J represents --NHSO₂ CH₃ or ##STR12##
 2. The compound ofclaim 1 wherein the compound is ##STR13##